key: cord-1017596-zwpgvn4y authors: Dennis Amoah, Isaac; Kumari, Sheena; Bux, Faizal title: Coronaviruses in wastewater processes: source, fate and potential risks date: 2020-07-09 journal: Environ Int DOI: 10.1016/j.envint.2020.105962 sha: 417eb7fbe400754ff4007dd4d87d519076348eb0 doc_id: 1017596 cord_uid: zwpgvn4y Abstract The last 17 years have seen three major outbreaks caused by coronaviruses, with the latest outbreak, COVID-19, declared a pandemic by the World Health Organization. The frequency of these outbreaks, their mortality and associated disruption to normal life calls for concerted efforts to understand their occurrence and fate in different environments. There is an increased interest in the occurrence of coronaviruses in wastewater from the perspective of wastewater-based epidemiology. However, there is no comprehensive review of the knowledge on coronavirus occurrence, fate and potential transmission in wastewater. This paper, provides a review of the literature on the occurrence of coronaviruses in wastewater treatment processes. We discuss the presence of viral RNA in feces as a result of gastrointestinal infections resulting in diarrhoea. We also review the literature on their presence, survival and potential removal in common wastewater treatment processes. The detection of infectious viral particles in feces of patients raises questions on the potential risks of infection for people exposed to untreated sewage/wastewater. We, therefore, discuss the potential risk of infection with coronaviruses for workers in wastewater treatment plants and the public that may be exposed through faulty plumbing or burst sewer networks. The disruption of life and mortalities warrants a much more focused research on the role of environments, such as wastewater and surface water, in disease transmission. The current wealth of knowledge on coronaviruses in wastewater based on the reviewed literature is scant and therefore calls for further studies. Coronaviruses may be introduced into wastewater (domestic and hospital) through several sources, such as handwashing, sputum and vomit Sung et al., 2016; Haagmans et al., 2014) . Additionally, there are reports of viral shedding in urine of individuals infected with SARS-CoV (Xu et al., 2005) , MERS (Drosten et al., 2013) and SARS-CoV-2 (Nomoto et al., 2020) . However, the main route that has been reported extensively is via the shedding of the viral RNA in feces of infected individuals (Ling et al., 2020; Xiao et al., 2020; Zhang et al., 2020b) . This section, therefore, focuses on the GI infections and reports of viral RNA in feces. Gastrointestinal infections (GI) symptoms, such as abdominal discomfort, diarrhea, GI bleeding, nausea and vomiting, have been observed in patients, indicating GI tract infection Wang et al., 2020a) . Studies have shown that the MERS-CoV infects and replicates in human primary intestinal epithelial cells, through the dipeptidyl peptidase receptor (van Doremalen et al., 2014; Wang et al., 2013) . In-vivo studies showed inflammation and epithelial degeneration in the small intestine before the development of pneumonia and brain infection associated with MERS-CoV (Zhou et al., 2017) . These findings suggest that in some MERS-CoV, pulmonary infections may be secondary to intestinal infections. Evidence suggests that the SARS-CoV and SARS-CoV-2 GI infections in humans are mediated through the angiotensin-converting enzyme 2 (ACE2) cell receptor (Yeo et al.,2020; Wan et al., 2020; Bertram et al., 2012; Chan et al., 2004b) . The ACE2 enzyme is mainly found attached to the cell membranes of cells in the lungs, arteries, heart, kidney, and intestines (Hamming et al., 2004) . The binding affinity of the ACE2 receptors has been observed as the most important factor for the infectivity of the virus (Yeo et al.,2020 , Holshue et al., 2020 . Structural analysis indicates that SARS-CoV-2 uses the human ACE2 receptor more efficiently than the SARS-CoV (Yeo et al.,2020) . This may be another reason for the faster spread of the SARS-CoV-2. Early reports from Wuhan showed that abdominal pain (an indication of GI infections) was reported more frequently in patients admitted into intensive care, than individuals not requiring intensive care Wang et al., 2020a; Chen et al., 2020a; Jin et al., 2020) . These reports also showed that in about 10% of the patients, diarrhoea and nausea symptoms occurred 1-2 days before the development of fever and respiratory symptoms . This again supports the hypothesis that in some patients, GI infections may occur before the respiratory symptoms. It is estimated that in the first SARS outbreak, between 20-25% of the patients had diarrhoea (Hui et al., 2003) , a sign of GI infections. Other publications have reported higher diarrhoea incidence among infected people, for instance 20.3%-38.4% (Leung et al., 2003) and 73% (Peiris et al., 2003) of patients are reported to have watery dirahhoea. In the current pandemic , reports on frequency of GI infections varies. For instance, Huang et al., (2020b) reported 3% diarrhoeal frequency. Wang et al., (2020a) reported GI infections like diarrhea (10.1%), nausea (10.1%), vomit (3.6%), abdominal pain (2.2%). However, official data from the WHO reports that between 2-27% of COVID-19 patients have diarrhoea (WHO, 2020b) . The detection of coronavirus in the feces of infected persons is therefore not surprising, based on the GI infections reported. Corman et al., (2016) reported MERS-CoV RNA in 14.6% of stool samples from patients. There are emerging reports of SARS-CoV-2 RNA in stool from patients in the current COVID-19 pandemic (Amirian, 2020; CDC, 2020a) . Table 1 presents reports of coronavirus RNA in stool samples from different geographical locations. However, it is unclear how long the shedding continues, a few studies have suggested that RNA can be found in the stool from day 1-25 days after onset of the GI illness (Amirian , 2020; Ling et al., 2020; The COVID-19 Investigation Team, 2020; Xiao et al., 2020; Zhang et al., 2020b) . In another study, SARS-CoV RNA was detected in stool samples from the fifth day, with a peak in viral titer at the 11 th day and lasted till the 30 th day (Amirian, 2020; Bell et al., 2003) . It is also unclear if there is any association between the detection of the viral RNA in stool and the severity or pattern of symptomatology of the disease (Amirian, 2020). However, it is assumed that both symptomatic and asymptomatic people could spread the virus through their excreta/feces (Núñez-Delgado, 2020) . In a study conducted in Wuhan, it was demonstrated that in about 10% of COVID-19 patients, viral RNA (some infectious) were still found in the feces even after no viral RNA was found in samples from the respiratory tract (Xiao et al., 2020) . Detection of the virus in fecal samples has been mainly through RT-PCR, as shown in Table 1 . This approach is the gold standard accepted globally for the detection of viral RNA in several types of clinical samples, however a stool sample positive for the virus may only have the RNA but not the infective viable virus. A few methods have used cell cultures (Chan et al., 2004a; Xu et al., 2005; Corman et al., 2016) , electron microscopy (Xu et al., 2005; Zhang et al., 2020a) and viral nucleocapsid staining (Xiao et al., 2020) for the detection of these viruses in feces. These approaches have shown that some fecal samples may contain viable viruses (Chan et al., 2004a; Xu et al., 2005; Zhang et al., 2020a) , which raises concern for transmission of infections through exposure to feces. The early onset of GI symptoms during infections with coronaviruses could therefore serve as an early warning system, based on early detection of the viral RNA in feces. Furthermore, continuous shedding of the viral RNA in feces after no respiratory symptoms are observed could potentially cause fecal-oral transmission from 'recovered' patients. Majority of studies on the occurence of viruses in wastewater have focused on nonenveloped enteric viruses, like adenoviruses, polio viruses, enteroviruses, noroviruses and rotaviruses (Ye et al., 2016; Fumian et al., 2010; Katayama et al., 2008) . This is mainly because these are transmitted primarily through the fecal-oral route (Ye et al., 2016) . However, the presence of enveloped viruses like coronaviruses in wastewater could differ greatly due to differences in their survival and partitioning behaviour in water (Ye et al., 2016; Arbely et al., 2006) . Since the cluster of SARS cases in an apartment block in Hong Kong, traced to droplets containing coronavirus from the wastewater system (WHO, 2003) , there has been an increased interest in the detection of coronaviruses in wastewater. Initial reports of SARS-CoV RNA in wastewater came from studies conducted at the Xiao Tang Shan Hospital and 309 th Hospital of PLA, the designated hospitals to receive SARS patients in Beijing during the 2003 outbreak (Wang et al., 2005c) . Another reason for increased interest in the occurrence of coronaviruses in wastewater is wastewater-based epidemiology (WBE). This concept aims to use sewage/untreated wastewater analysis as an early warning system for disease outbreak (Xagoraraki & O'Brien, 2020) , since viral RNA can be detected in feces, and subsequently wastewater, weeks before the onset of illness. A few studies have reported the detection of coronavirus in untreated wastewater/sewage (Table 2 ). These studies have focused mainly on the detection of these viruses without quantification, therefore it challenging to compare the concentration of the viral titer between studies. The available information shows an increase in these studies during the current pandemic, this could be attributed to the WBE concept,the need for further information on the occurrence of these viruses in wastewater and the avaibility of advanced molecular techniques for viral load quantification. In Paris, Wurtzer et al., (2020) detected the presence of the SARS-CoV-2 viral RNA even in the treated wastewater. However, the presence of the viral RNA does not indicate that these viral particles are intact and infectious. Addtionally, in the report the wastewater treatment processes used was not stated. The main factor influencing the occurrence of coronaviruses in wastewater will be the concentration of viral RNA shed per gram of feces of an infected person. In general, the concentration of enteric viral particles per gram of feces during diarrhoea has been reported to be 10 10 -10 12 (Haas et al., 2014) . For SARS viral loads of 10 6.1 gc/g of feces and 10 1.3 gc/mL of urine have been reported (Hung et al., 2004) . Reports of viral load in stool of persons infected with SARS-CoV-2 has varied. For instance, 1.7 × 10 6 -4.1 × 10 7 gc/mL have been reported by Han et al., (2020b) , in contrast to 6.3× 10 6 -1.26× 10 8 gc/g of stool reported by Lescure et al., (2020) . In anal swabs viral loads of 10 5 gc/swab for SARS-CoV-2 has been reported (Woelfel et al., 2020) . This shows that the viral load of coronaviruses in feces may be lower than that of enteric viruses. However, additional studies are required to understand how frequently coronaviruses are shed in feces and urine of infected individuals. This information will not only give an idea to the concentrations expected in wastewater but may also provide vital information in understanding the potential of fecal transmission. Information on the shedding frequency will also aid in correlating the viral load in the wastewater with infection rate in the community. In addition, the per capita water use could affect the concentration of viruses detected in wastewater. Peak times (like mornings and evenings) are associated with higher domestic water usage Almeida et al., 1999) , this could result in dilution, therefore, reducing the concentrations of viral load at these times. The survival of these coronaviruses in the environment could be another major factor influencing their occurrence in wastewater. The current belief is that coronaviruses can survive for only a few days in the environment (Kampf et al., 2020) , however, some studies paint a different picture, this is discussed in detail in Section 4.1. Detection of coronavirus RNA in wastewater has been mainly through molecular techniques involving PCR based methods such as reverse transcription-polymerase chain reaction (RT-PCR) and digital PCR. This is achieved through the amplification of parts of the viral genome, like the genes coding for either the nucleocapsid (CDC, 2020b), and viral envelop . Molecular detection of the viral RNA involves three major steps. These include;  Viral concentration/enrichment: Due to the potential low concentration of viral titer in wastewater, several options have been used to concentrate the viral particles for analysis. These include; direct analysis of unfiltered wastewater/sewage samples after precipitation with polyethylene glycol (PEG) . Viral concentration/enrichment through filtration using 0.2um filters , Amplification of viral RNA: Amplification of viral RNA extracted from wastewater has been performed with a set of five primers/probes. These primers and probes target different parts of the viral particle as shown in Table 3 . Varying results have been reported using these primer/probe sets targeting different parts of the viral genome. For instance, Medema et al., (2020) observed positive amplification from all study sites (6) using the N1 primer, as compared to the N3 and E primers that were positive in 5 and 4 study sites respectively. However, in contrast Rimoldi et al., (2020) reported a high frequency of positive amplification targeting the ORF1ab gene , compared with the N and E genes that were only positive in three of the positive wastewater samples. These results therefore indicate inconclusive results in relation to the best primer/probe set for amplification of the viral RNA in wastewater . This could be attributed to primer/probe sensitivity, PCR inhibitors in the wastewater sampels from different regions/sites and potential stability of the virus and viral genome in these different areas. 14 | P a g e Cor Arora et al., 2020; Kumar et al., 2020; Rimoldi et al., (2020 Other molecular methods have been used for the detection of coronavirus RNA in clinical samples, such as pharyngeal swabs, these include reverse transcription (RT) loop-mediated isothermal amplification (LAMP) (Park et al., 2020a; Lamb et al., 2020; Yu et al., 2020; Huang et al., 2020a; Shirato et al., 2018; Li et al., 2015; Shirato et al., 2014) . This method has shown great potential in detecting these viruses in clinical samples, producing results in less than an hour, in some instances within 11 minutes (Thai et al., 2004) . Another molecular technique used for coronavirus detection in clinical and wastewater samples is the digital droplet PCR. This has shown to be have an improved lower limit of detection, more sensitive and accurate compared to RT-PCR for environmental samples Dong et al., 2020; . The use of dPCR may therefore aid in reducing false negatives and positives, especially in samples with low viral titer, like wastewater . The molecular detection and quantification of viral RNA in wastewater using these molecular techniques has shown the potential for the use of wastewater analysis to determine the infection Queensland, Australia. In addition to the estimation of infected people through the accurate detection and quantification of viral load, this approach could also act as an early warning system. This is possible due to the early shedding (1-2 days) of viral RNA in the stool of a proportion of infected individuals before the onset of pulmonary symptoms . For instance, Medema et al., (2020) detected SARS-CoV-2 viral RNA in wastewater taken almost a week (6 days) before the first case of COVID-19 was reported in the city of Amersfoort in the Netherlands. Human enveloped viruses, like coronaviruses, are presumed to undergo rapid inactivation in the water environment (Kampf et al., 2020; Ye et al., 2016) . However, several reports of these viruses in feces and wastewater, as discussed in the sections above, indicate these may be able to survive longer than presumed. The fate of coronaviruses in wastewater may be mediated by two processes; their ability to survive in the extreme wastewater environment and its removal during different stages of wastewater treatment. In this section, we review the current knowledge surrounding coronavirus survival in wastewater and discuss their possible removal by different wastewater treatment processes. There is currently evidence to suggest that SARS-CoV and MERS-CoV are viable under different environmental conditions. Gundy et al., (2009) reported that it will take 2-3 days for a 99.9% reduction of coronavirus in wastewater, this agrees with data from Wang et al (2005b) at 20 0 C. In unpasteurized wastewater Ye et al., (2016) , observed that it takes 13(±1) hours for 90% inactivation. In contrast, Casanova et al., (2009) , reported that it could take up to a week for coronaviruses in wastewater to reduce by 99%. This data was generated using the mouse hepatitis virus (MHV) and transmissible gastroenteritis virus (TGEV) as surrogates for coronaviruses. The experiments were also performed using pasteurized water, which could have eliminated the possible predation action from other microbes in the wastewater. Additionally, the longer survival of the surrogates, MHV and TGEV, as compared to the human coronaviruses, SARS-CoV and MERS-CoV, could be attributed to the subtle difference between these different viruses. It can be concluded, based on the data available, that it could take a maximum of 3 days for 99.9% or a 3 Log reduction of coronaviruses in wastewater at 20°C. This could, however, be affected greatly by several factors discussed in Section 4.1.1. Infectivity of coronaviruses in wastewater could also be affected. The study by Casanova et al., (2009) , indicated that it takes 7-9 days for a 99% reduction in infectious viral titer at 25°C. Additionally, SARS-CoV seeded into sewage remained infectious for 2 days at 20°C (Yeo et al., 2020; Wang et al., 2005b) . Reports on the occurrence of SARS-CoV-2 in wastewater is mainly based on nucleic acid (RNA) detection . Rimoldi et al., (2020) reported no viable SARS-CoV-2 in wastewater based on cell cultures. They estimated that it took 6-8 hours from excretion in feces to the wastewater sampling point, therefore the virus may have been inactivated within that period. However, based on data from other coronaviruses (as discussed above) and occurrence of viable SARS-CoV-2 viral particles in feces (Chan et al., 2004a; Xu et al., 2005; Zhang et al., 2020a) , untreated wastewater may contain some viable and infective human coronaviruses. Survival for MHV to achieve the same inactivation at the same conditions (Casanova et al., 2009 ). The faster inactivation of coronaviruses in wastewater could be attributed to the presence of chemicals with antiviral activity (Sobsey & Meschke, 2003) , proteolytic enzymes produced by bacteria (Casanova et al., 2009) , protozoan and metazoan predation in the wastewater (Ye et al., 2016) . Additionally, survival studies on viruses in wastewater have found that the high molecular weight of dissolved matter, which is common wastewater, may influence their survival (Noble & Fuhrman, 1997) .  Temperature: Just like many other microorganisms' temperature has been found to have a greater influence on the survival of coronaviruses in wastewater. Using SARS-CoV seeded into sewage, it was observed that the viruses remain infectious for 14 days at 4°C, but for only 2 days at 20°C (Wang et al., 2005c) . Studies on the survival of TGEV and MHV also showed that at 25°C it took 19 days for TGEV and 14 days to reduce by 4 log10 in wastewater. However, at 4°C for the same level of reduction, it will take 98 days for TGEV and 139 days for MHV (Casanova et al., 2009)  pH: There is a lack of information on the impact of pH on the survival of coronaviruses. However, based on the stability of MHV and TGEV we can deduce the impact of wastewater pH on coronaviruses survival. A pH range of 5-7.4 at 37°C and 3-10 at 4°C is considered to be the stable range for MHV (Casanova et al., 2009; Daniel & Talbot, 1987) . For TGEV, the stable pH range is 5-7 at 37°C and 5-8 at 4°C. Acidic pH has been shown to result in reversible acid denaturation of RNA, through protonation of GC base pairs and consequent formation of Hoogsteen base pairing (Mariani, et al., 2018) . In addition to the impact on viral stability, pH influences viral survival through the impact on adsorption on particles in the wastewater. An increase in viral adsorption is observed with decreasing pH (Schaub et al., 2017) . Conventional wastewater treatment processes are mainly designed for the removal of organic matter and suspended solids (Droste & Gehr, 2018; Qasim, 2017) . Some degree of pathogen removal is however achieved in the process, but this is mainly effective for bacteria than viruses (Dias et al., 2018; Diston et al., 2012; Sinton et al., 2002; Grabow, 2001) . Several studies have reported the removal efficiency of enteric viruses during wastewater treatment; however, only one study has reported coronavirus removal during wastewater treatment. Wurtzer et al., (2020) detected SARS-CoV-2 RNA in treated and untreated wastewater from Paris, concentrations in the treated wastewater were found to be 100 times lower than viral load in the untreated wastewater. However, this study did not report the type of wastewater treatment processes employed in these treatments, nor the viability or otherwise of these viral particles. The viral RNA detected in the treated wastewater could also be pieces of the viral particle, therefore this information is inconclusive in helping to understand the possible removal of coronaviruses during wastewater treatment. Viral adsorption and inactivation have been given as the two main reasons for reduction in water (Bibby et al., 2015) . This has been observed for Φ6 bacteriophage, which is also an enveloped RNA virus (Bibby Simmons Nordgren et al., 2009 ). This treatment process includes primary settling, biological degradation and secondary clarification (Sidhu et al., 2017; Keegan et al., 2013) . At equilibrium in wastewater, Ye et al., (2016) demonstrated that MHV (used as a surrogate for human coronaviruses) adsorbs to the wastewater solids more rapidly. They estimated that 26% of the MHV will be adsorbed to the wastewater solids at equilibrium, with 99% equilibrium occurring at 0.4-2.9 h. Therefore, it can be postulated that during ASP processes the highest removal of coronaviruses may occur at the primary settling stage. It has been reported that microbial inactivation increases with increasing hydraulic retention time (HRT), till a saturation is reached (Garcıá, et al., 2003) . For instance, in a wastewater pond system, Verbyla and Mihelcic, (2015) reported an average of 1 log10 reduction of viruses for every 14.5-20.9 days of retention. Therefore, in addition to adsorption to solids, a longer HRT may also be critical in inactivating coronaviruses in wastewater. The adsorption of coronaviruses to the solids therefore means a high concentration may be expected in the sludge. Anaerobic digestion of sludge, which is a common sludge treatment process, has proven to result in reduction of pathogenic microorganisms. Sassi et al., (2018) demonstrated that mesophilic anaerobic digestion could achieve above 5.9 log 10 reduction of the Φ6 bacteriophage. This could be attributed to protein and nucleic acid denaturation at higher temperatures. We can there conclude that coronavirus adsorbed on the wastewater solids could be effectively removed (almost by 6 Log10 units) during anaerobic digestion. Viral removal between 2-3 log10 has been reported for different types of viruses during membrane bioreactor (MBR) treatment (Prado et al., 2019; Miura et al., 2018; Purnell et al., 2016) . Other studies have reported Log removal of viral particles greater than 4 (Chaudhry et al., 2015; Kuo et al., 2010) . The main mechanism in the MBR processes responsible for viral, and other pathogen removals, is retention or size exclusion. Chaudhry et al. (2015) reported that retention by a 0.04 µm membrane accounts for over 50% removal of in use (Nqombolo et al., 2018) . With an average viral particle diameter of 120 nm (.12 μm) and envelop diameter of 80 nm (.08 μm) (Neuman & Buchmeier, 2016) coronaviruses, the best membrane technology for their removal will be ultrafiltration. The adsorption of coronaviruses to solids in wastewater may enhance their removal. Tertiary wastewater treatment processes such chlorination and UV treatment may also result in further removal of remaining coronaviruses in the wastewater. Wang et al., (2005b) reported that SARS-CoV can be inactivated completely by 20 mg/L chlorine in 1 min. They observed that Chlorine dioxide was less effective for the inactivation of SARS-CoV as compared with free chlorine, similar results have been reported for other viruses (Young et al., 2020b; Wati et al., 2019; Cromeans et al., 2010; Lim et al., 2010) . Chlorine has been reported to inactivate viruses through the cleavage of the capsid protein backbone of viruses, therefore inhibiting viral genome injection into host cells Page et al., 2010) . Several studies have also reported the inactivation of coronaviruses using UV irradiation (Shirbandi et al., 2020; Kim & Jang, 2018; Casanova et al., 2009; Wang et al., 2005b) . Pinon and Vialette (2018) reported that enveloped viruses, like coronaviruses are more sensitive to UV than non-enveloped viruses. The main mechanism through which UV inactivates coronaviruses could be through the generation of pyrimidine dimers which damages the nucleic acid (Smith & Denison, 2013) . It must be noted that in addition to the specific wastewater treatment processes that may result in the removal or inactivation of coronaviruses, factors affecting their survival in wastewater (Section 4.1.1) may also contribute significantly. The occurrence of infective viral particles of coronaviruses in wastewater may pose health concerns for people who come into contact with the wastewater. Live SARS-CoV-2 has been isolated from stools of patients Wang et al., 2020b; Xiao et al., 2020) , in one instance 15 days after onset of the disease . With the observation that it may take 2 days for a 99% reduction in infectivity (Section 4.1), it is safe to assume that some of the viral particles may still be infectious. The present belief is that SARS-CoV-2 has a low infectious dose (Lee & Hsueh, 2020) , therefore the viral loads in the wastewater could still pose a great risk. The available information on the viral survival implies that the populations at greatest risks are people exposed to the raw sewage. This could be workers at wastewater treatment plants (WWTPs) and the general public who may be directly exposed to the sewage via faulty plumbing or sewer networks. Despite these fears till date there is no evidence for the transmission of COVID-19 due to exposure to wastewater (WHO, 2020b). There is evidence to suggest that GI infections may occur first in a subset of coronavirus infections (Section 3.1), which means that workers at WWTPs may be exposed to these pathogens days before an outbreak is reported. Exposure within the WWTPs could be through two major routes; aerosol inhalation or direct contact with infectious viral particles. Inhalation of aerosols or droplets contaminated with infectious viral particles has been reported as the main route through which coronaviruses are transmitted in WWTPs ( C. Viral inactivation: This is expressed as a function of time and meteorological factors, such as temperature and humidity (Zhao et al., 2014) . At 25 0 C and relative humidity (RH) of 79%, over 60% of coronaviruses in aerosols/droplets have been found to remain infectious for up to 60 minutes, however at much warmer temperature, of 38 °C and 24% RH, only 4.7% remained infectious (Pyankov et al., 2018) . Therefore, it is safe to assume that within an hour aerosol contaminated with coronaviruses will still contain some number of infectious particles that may result in infections. D. Amount of infectious viral particle inhaled: The breathing rate, lung volume and Bonow et al., 2020; Garg, 2020) . However, healthy individuals are also infected upon exposure, therefore all workers within the WWTPs irrespective of their health status may be at risk of infection especially when surface aeration is used as compared to diffused aeration. Beyond direct inhalation of aerosols in the WWTPs, direct contact with the infectious viral particles deposited on contact surfaces may also result in infections. The potential for aerosols contaminated with infectious viral particles been deposited on contact surfaces/formites is high within the WWTPs. The detection of coronaviruses in environmental samples, especially in nosocomial infections (Xiao et al., 2017; Booth et al., 2005) and evidence that intranasal instillation could cause infections (Xiao et al., 2017) shows that direct contact with the viral particles on formites may be a major route of transmission. The findings that handwashing reduces the risks of infections with these viruses further supports the role of direct hand contact in the transmission (Xiao et al., 2017; Lau et al., 2004; Teleman et al., 2004) . Analysis of studies on the survival of coronaviruses on surfaces, showed that these infectious viral particles can survive up to nine days at room temperature (Kampf et al., 2020) . However, they can be easily inactivated within a minute using 62-71% ethanol, 0.5% hydrogen peroxide or 0.1% sodium hypochlorite. In addition to viral particles deposited on formites/contact surfaces within the WWTP, direct exposure to the viral particles may occur during removal and transportation of primary and secondary sludge. Some WWTPs may not have facilities for onsite sludge treatment, which requires the collection and transportation of the sludge for treatment off-site. Occurrence of human coronaviruses been reported in sludge both treated (Bibby et al., 2011; Bibby& Peccia, 2013 ) and untreated sludge (Bibby& Peccia, 2013) . This could therefore be another significant exposure route for workers of the WWTPs. For instance, Westrell et al., (2004) reported a high risk of viral infection for workers during sludge dewatering. Therefore, without proper hygienic practices and use of PPE, WWTP workers could be exposed to infectious coronavirus particles deposited on surfaces and in sludge. The case of SARS infections from the apartment block in Hong Kong, highlighted the role of the wastewater system or sewer networks in the spread of infections (WHO, 2003) . This was spread through droplets from SARS-CoV contaminated sewage from a faulty plumbing. It therefore shows that exposure to the sewage containing infectious coronaviruses may lead to the spread of the virus. In addition to faulty plumbing within residential facilities, burst sewer networks discharging untreated wastewater into the community may be another route of transmission. Faults in sewer networks, either structural or hydraulic, may take a long time before repairs (Khan et al., 2009) , which could increase the risks. Areas with inadequate sanitation, which means no sewer networks, will have higher risks of exposure to the coronavirus in the sewage. Exposure to the untreated sewage in these circumstances may pose higher risks than the risks exposed to the workers at the WWTPs. This is because this exposure may occur minutes or hours after excretion of the viral particles where infectivity may still be high.Furthermore, the discharge of treated wastewater directly into surface water bodies could potentially lead to exposure for the general public. This is especially critical in areas where WWTPs are not working effectively resulting in occurrence of pathogens in the final effluent. Occurrence of coronaviruses in surface water has been reported in Saudi Arabia (Blanco et al., 2019) and Kazakstan (Alexyuk et al., 2017) . Rimoldi et al (2020) also detected SARS-CoV-2 RNA in surface water which they attributed this occurrence to discharge of non-collected domestice wastes or urban runoff from domestic effluents. However, no viable viral particle was founf based on cell cultures, which indicates the potential absence of risks of infections from surface water. Further work is required to fully assess the possible risks from coronaviruses in wastewater. Future studies in this regard could focus on; 1. The shedding frequency of coronavirus RNA in feces and urine: This is necessary in understanding the viral load in the wastewater per an infected person or population. wastewater: There is the need for more research on the effective recovery of coronavirus from wastewater and optimization of the RNA extraction methods. Additionally, the different primers used for the amplification of the viral particles could introduce uncertainties into the results due to difference in stability of the various parts of the viral particle. Therefore, there is the need for research to understand how stable the different viral particles are in wastewater. Arora, S., Nag, A., Sethi, J., Rajvanshi, J., Saxena, S., Shrivastava, S.K. and Gupta, A.B., 2020. Sewage surveillance for the presence of SARS-CoV-2 genome as a useful wastewaterbased epidemiology (WBE) tracking tool in India. medRxiv.  Coronavirus infections may lead to gastrointestinal symptoms such as diarrhoea, resulting in the shedding of viral particles in feces and urine.  The viral shedding leads to their occurrence in wastewater and may remain infectious for up to 2 days at 20°C.  Over 26% of coronaviruses may adsorb to solid particles in wastewater, however conventional wastewater treatment processes can remove or inactivate these viruses.  The presence of infectious viral particles in wastewater up to 2 days, may expose the general public, wastewater treatment plant workers to possible risk of infections GRAPHICAL ABSTRACT: A graphical representation of the source and fate of Coronavirus in wastewater, showing the potential viral load in feces and urine and their survival removal by during wastewater/sludge treatment. The potential exposure points are represented by the letter 'E' in a red background. These exposure points include; the home setting due to faulty plumbing, general public exposure to untreated sewage due to inadequate sanitation and burst sewer pipes. Exposure for WWTW workers from aerosols and direct contact due to sludge collection and treatment. The final exposure scenario is for the general public due to the potential presence of infectious viral particles in treated sewage. Association of coronavirus disease 2019 (covid-19) with myocardial injury and mortality Detection of airborne severe acute respiratory syndrome (SARS) coronavirus and environmental contamination in SARS outbreak units Coronavirus May Cause Environmental Contamination Through Fecal Shedding Human viral pathogens are pervasive in wastewater treatment center aerosols A Case Series of children with 2019 novel coronavirus infection: clinical and epidemiological features Survival of surrogate coronaviruses in water CDC-Centers for Disease Control & Prevention. 2020a. Checklist for Healthcare Facilities: Strategies for Optimizing the Supply of N95 Respirators during the COVID-19 Real-Time RT-PCR Diagnostic Panel A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster Detection of SARS coronavirus in patients with suspected SARS Persistent infection of SARS coronavirus in colonic cells in vitro Mechanisms of pathogenic virus removal in a full-scale membrane bioreactor. Environmental science & technology Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study Structure analysis of the receptor binding of 2019-nCoV. Biochemical and biophysical research communications The presence of SARS-CoV-2 RNA in the feces of COVID-19 patients Viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR Viral shedding and antibody response in 37 patients with Middle East respiratory syndrome coronavirus infection A new solid matrix for preservation of viral nucleic acid from clinical specimens at ambient temperature Physico-chemical properties of murine hepatitis virus, strain A 59 The application of bacteriophages as novel indicators of viral pathogens in wastewater treatment systems The effect of UV-C radiation (254 nm) on candidate microbial source tracking phages infecting a human-specific strain of Bacteroides fragilis (GB-124) Interlaboratory assessment of droplet digital PCR for quantification of BRAF V600E mutation using a novel DNA reference material Theory and practice of water and wastewater treatment Clinical features and virological analysis of a case of Middle East respiratory syndrome coronavirus infection. The Lancet infectious diseases Detection of rotavirus A in sewage samples using multiplex qPCR and an evaluation of the ultracentrifugation and adsorption-elution methods for virus concentration Morphological and functional implications of sexual dimorphism in the human skeletal thorax Role of hydraulic retention time and granular medium in microbial removal in tertiary treatment reed beds Hospitalization Rates and Characteristics of Patients Hospitalized with Laboratory-Confirmed Coronavirus Disease 2019-COVID-NET, 14 States Possible aerosol transmission of COVID-19 and special precautions in dentistry How much reduction of virus is needed for recycled water: A continuous changing need for assessment? Water research Bacteriophages: update on application as models for viruses in water Quantification of SARS-CoV-2 and cross-assembly phage (crAssphage) from wastewater to monitor coronavirus transmission within communities Clinical characteristics of coronavirus disease 2019 in China Survival of coronaviruses in water and wastewater Middle East respiratory syndrome coronavirus in dromedary camels: an outbreak investigation. The Lancet infectious diseases Microbial agents and transmission Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis SARS-CoV-2 RNA more readily detected in induced sputum than in throat swabs of convalescent COVID-19 patients. The Lancet Infectious Diseases Sequential analysis of viral load in a neonate and her mother infected with SARS-CoV-2 First environmental surveillance for the presence of SARS-CoV-2 RNA in wastewater and river water in Japan Detection of SARS-CoV-2 in wastewater in Japan by multiple molecular assays-implication for wastewater-based epidemiology (WBE) Middle East respiratory syndrome coronavirus and the one health concept Viral evolution: variation in the gut virome First case of 2019 novel coronavirus in the United States 2020a. RT-LAMP for rapid diagnosis of coronavirus SARS-CoV-2. Microbial Biotechnology Clinical features of patients infected with 2019 novel coronavirus in Wuhan SARS: clinical features and diagnosis Viral loads in clinical specimens and SARS manifestations Epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019 (COVID-19) with gastrointestinal symptoms A Well Infant with Coronavirus Disease 2019 with High Viral Load Persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents One-year monthly quantitative survey of noroviruses, enteroviruses, and adenoviruses in wastewater collected from six plants in Japan Validation of activated sludge plant performance for virus and protozoan reduction Stochastic Analysis of Factors Affecting Sewer Network Operational Condition Inactivation of airborne viruses using vacuum ultraviolet photocatalysis for a flow-through indoor air purifier with short irradiation time Relative abundance and treatment reduction of viruses during wastewater treatment processes-identification of potential viral indicators SARS-CoV-2 Detection in Istanbul Wastewater Treatment Plant Sludges The first proof of the capability of wastewater surveillance for COVID-19 in India through the detection of the genetic material of Assessment of human adenovirus removal in a full-scale membrane bioreactor treating municipal wastewater Rapid Detection of Novel Coronavirus (COVID19) by Reverse Transcription-Loop-Mediated Isothermal Amplification First detection of SARS-CoV-2 in untreated wastewaters in Italy Coronaviruses: emerging and re-emerging pathogens in humans and animals SARS transmission, risk factors, and prevention in Hong Kong Emerging threats from zoonotic coronaviruses-from SARS and MERS to 2019-nCoV Minimum Size of Respiratory Droplets Containing SARS-CoV-2 and Aerosol Transmission Possibility Clinical and virological data of the first cases of COVID-19 in Europe: a case series Enteric involvement of severe acute respiratory syndrome-associated coronavirus infection Detection of middle east respiratory syndrome coronavirus by reverse-transcription loop-mediated isothermal amplification SARS-CoV-2 detection using digital PCR for COVID-19 diagnosis, treatment monitoring and criteria for discharge Disinfection kinetics of murine norovirus using chlorine and chlorine dioxide Aerosolization of Ebola virus surrogates in wastewater systems The persistence and clearance of viral RNA in 2019 novel coronavirus disease survivors The value of urine biochemical parameters in the prediction of the severity of coronavirus disease 2019 Evaluation of SARS-CoV-2 RNA shedding in clinical specimens and clinical characteristics of 10 patients with COVID-19 in Macau SARS-CoV-2 in wastewater: potential health risk, but also data source pH-Driven RNA strand separation under prebiotically plausible conditions The genome sequence of the SARS-associated coronavirus Presence of SARS-Coronavirus-2 in sewage Virus typespecific removal in a full-scale membrane bioreactor treatment process Coronaviruses Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater Supramolecular architecture of the coronavirus particle Virus decay and its causes in coastal waters Cautious handling of urine from moderate to severe COVID-19 patients Prevalence of norovirus and factors influencing virus concentrations during one year in a full-scale wastewater treatment plant Wastewater treatment using membrane technology What do we know about the SARS-CoV-2 coronavirus in the environment? Science of The Total Environment Regressing SARS-CoV-2 sewage measurements onto COVID-19 burden in the population: a proof-of Mechanistic aspects of adenovirus serotype 2 inactivation with free chlorine Viral load of SARS-CoV-2 in clinical samples. The Lancet Infectious Diseases Development of Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assays Targeting SARS-CoV-2 Detection of SARS-CoV-2 in Fecal Samples from Patients with Asymptomatic and Mild COVID-19 in Korea SARS-CoV-2 RNA concentrations in primary municipal sewage sludge as a leading indicator of COVID-19 outbreak dynamics Coronavirus as a possible cause of severe acute respiratory syndrome Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study Survival of viruses in water Kinetics and pattern of viral excretion in biological specimens of two MERS-CoV cases Noroviruses in raw sewage, secondary effluents and reclaimed water produced by sand-anthracite filters and membrane bioreactor/reverse osmosis system Removal of phages and viral pathogens in a full-scale MBR: implications for wastewater reuse and potable water Wastewater treatment plants: planning, design, and operation Wastewater treatment plants: planning, design, and operation Retrospective Search for SARS-CoV-2 in Human Faecal Metagenomes SARS-CoV-2 RNA in wastewater anticipated COVID-19 occurrence in a low prevalence area Presence and vitality of SARS-CoV-2 virus in wastewaters and rivers. medRxiv Computational modeling of aerosol deposition in respiratory tract: a review Comparative survival of viruses during thermophilic and mesophilic anaerobic digestion Physics and Modelling of Wind Erosion-Front Matter. Physics and Modelling of Wind Erosion Detection of SARS-Coronavirus-2 in wastewater, using the existing environmental surveillance network: An epidemiological gateway to an early warning for COVID-19 Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus Detection of Middle East respiratory syndrome coronavirus using reverse transcription loop-mediated isothermal amplification (RT-LAMP) Inactivation of Coronavirus with Ultraviolet Irradiation: What? How? Why? Comparative enteric viruses and coliphage removal during wastewater treatment processes in a sub-tropical environment Release of infectious human enteric viruses by fullscale wastewater utilities Removal of human enteric viruses by a full-scale membrane bioreactor during municipal wastewater processing Sunlight inactivation of fecal indicator bacteria and bacteriophages from waste stabilization pond effluent in fresh and saline waters Coronaviruses as DNA wannabes: a new model for the regulation of RNA virus replication fidelity Virus survival in the environment with special attention to survival in sewage droplets and other environmental media of fecal or respiratory origin. Report for the World Health Organization Epidemiology, genetic recombination, and pathogenesis of coronaviruses Comparative evaluation of three homogenization methods for isolating Middle East respiratory syndrome coronavirus nucleic acids from sputum samples for real-time reverse transcription PCR Early Release-Detection of Novel Coronavirus by RT-PCR in Stool Specimen from Asymptomatic Child First 12 patients with coronavirus disease 2019 (COVID-19) in the United States Factors associated with transmission of severe acute respiratory syndrome among health-care workers in Singapore Recognition of aerosol transmission of infectious agents: a commentary Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus Host species restriction of Middle East respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 Atmospheric dispersion modelling of bioaerosols that are pathogenic to humans and livestock-A review to inform risk assessment studies A review of virus removal in wastewater treatment pond systems Source apportionment of particulate matter in Europe: a review of methods and results Receptor recognition by the novel coronavirus from Wuhan: an analysis based on decade-long structural studies of SARS coronavirus Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in Wuhan, China Detection of SARS-CoV-2 in different types of clinical specimens Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4 Excretion and detection of SARS coronavirus and its nucleic acid from digestive system Study on the resistance of severe acute respiratory syndrome-associated coronavirus Concentration and detection of SARS coronavirus in sewage from Xiao Tang Shan Hospital and the 309th Hospital of the Chinese People's Liberation Army. Water science and technology Chlorine inactivation of coxsackievirus B5 in recycled water destined for non-potable reuse QMRA (quantitative microbial risk assessment) and HACCP (hazard analysis and critical control points) for management of pathogens in wastewater and sewage sludge treatment and reuse Middle East respiratory syndrome coronavirus (MERS-CoV) WHO-World Health Organization, 2020b. Water, sanitation, hygiene and waste management for COVID-19: technical brief Coronavirus disease (COVID-19) Situation Report -166 WHO-World Health Organization Regional Office for the Western Pacific Virus disinfection mechanisms: the role of virus composition, structure, and function. Current opinion in virology Virus inactivation mechanisms: impact of disinfectants on virus function and structural integrity Can we contain the COVID-19 outbreak with the same measures as for SARS Asymptomatic SARS coronavirus infection among healthcare workers Modelling the effect of size on the aerial dispersal of microorganisms Clinical presentation and virological assessment of hospitalized cases of coronavirus disease Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia SARS-CoV-2 titers in wastewater are higher than expected from clinically confirmed cases Risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease Prolonged presence of SARS-CoV-2 viral RNA in faecal samples Time course quantitative detection of SARS-CoV-2 in Parisian wastewaters correlates with COVID-19 confirmed cases Wastewater-Based Epidemiology for Early Detection of Viral Outbreaks Evidence for gastrointestinal infection of SARS-CoV-2 Role of fomites in SARS transmission during the largest hospital outbreak in Hong Kong Comparison of different samples for 2019 novel coronavirus detection by nucleic acid amplification tests Prolonged presence of SARS-CoV-2 in feces of pediatric patients during the convalescent phase Persistent shedding of viable SARS-CoV in urine and stool of SARS patients during the convalescent phase Prevalence of comorbidities in the novel Wuhan coronavirus (COVID-19) infection: a systematic review and meta-analysis Survivability, partitioning, and recovery of enveloped viruses in untreated municipal wastewater Enteric involvement of coronaviruses: is faecal-oral transmission of SARS-CoV-2 possible Epidemiologic features and clinical course of patients infected with SARS-CoV-2 in Singapore Relationship Between Inactivation and Genome Damage of Human Enteroviruses Upon Treatment by UV 254, Free Chlorine, and Ozone Rapid detection of COVID-19 coronavirus using a reverse transcriptional loopmediated isothermal amplification (RT-LAMP) diagnostic platform Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia Isolation of 2019-nCoV from a stool specimen of a laboratoryconfirmed case of the coronavirus disease 2019 (COVID-19) Fecal specimen diagnosis 2019 novel coronavirusinfected pneumonia Detectable SARS-CoV-2 viral RNA in feces of three children during recovery period of COVID-19 pneumonia Airborne microorganisms from livestock production systems and their relation to dust Dynamic detection of RNA of SARSassociated coronavirus in blood and excreta of SARS patients Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province Human intestinal tract serves as an alternative infection route for Middle East respiratory syndrome coronavirus Potential transmission risk of SARS-CoV-2 through medical wastewater in COVID-19 outbreak cities A novel coronavirus from patients with pneumonia in China ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests